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Prevalence of antimicrobial resistance and resistancegenes in faecal isolates recovered from healthy pets
Daniela Costa, Patricia Poeta, Yolanda Sáenz, Ana Cláudia Coelho, ManuelaMatos, Laura Vinué, Jorge Rodrigues, Carmen Torres
To cite this version:Daniela Costa, Patricia Poeta, Yolanda Sáenz, Ana Cláudia Coelho, Manuela Matos, et al.. Prevalenceof antimicrobial resistance and resistance genes in faecal isolates recovered from healthy pets. Veteri-nary Microbiology, Elsevier, 2007, 127 (1-2), pp.97. �10.1016/j.vetmic.2007.08.004�. �hal-00532303�
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Accepted Manuscript
Title: Prevalence of antimicrobial resistance and resistancegenes in faecal Escherichia coli isolates recovered fromhealthy pets
Authors: Daniela Costa, Patricia Poeta, Yolanda Saenz, AnaClaudia Coelho, Manuela Matos, Laura Vinue, JorgeRodrigues, Carmen Torres
PII: S0378-1135(07)00392-6DOI: doi:10.1016/j.vetmic.2007.08.004Reference: VETMIC 3783
To appear in: VETMIC
Received date: 25-4-2007Revised date: 6-8-2007Accepted date: 7-8-2007
Please cite this article as: Costa, D., Poeta, P., Saenz, Y., Coelho, A.C., Matos, M., Vinue,L., Rodrigues, J., Torres, C., Prevalence of antimicrobial resistance and resistance genesin faecal Escherichia coli isolates recovered from healthy pets, Veterinary Microbiology(2007), doi:10.1016/j.vetmic.2007.08.004
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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Revised version1
Prevalence of antimicrobial resistance and resistance genes in faecal Escherichia 2
coli isolates recovered from healthy pets3
4
Daniela Costa1,4, Patricia Poeta1, 2, Yolanda Sáenz4, Ana Cláudia Coelho1, Manuela 5
Matos1,3, Laura Vinué4, Jorge Rodrigues1,2 and Carmen Torres4*6
1Universidade de Trás-os-Montes e Alto Douro; Departamento de Ciências 7
Veterinárias; Vila Real, Portugal; 2Centro de Estudos de Ciência Animal e Veterinária, 8
Vila Real, Portugal; 3Departamento de Genética e Biotecnología/Instituto de 9
Biotecnología e Bioengenharia, Vila Real, Portugal; 4Area de Bioquímica y Biología 10
Molecular, Universidad de La Rioja, Logroño, Spain11
12
Running title: antimicrobial resistance in faecal E. coli of pets13
Keywords: dogs, cats, Escherichia coli, antimicrobial resistance, CTX-M-1, OXA-3014
15
Corresponding author.16
Carmen Torres 17
Área de Bioquímica y Biología Molecular18
Universidad de La Rioja19
Madre de Dios, 5120
26006 Logroño, Spain21
FAX: 34-94129972122
Phone: 34-94129975023
e-mail: [email protected]
revised Manuscript
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Abstract 25
Faecal samples of healthy dogs (n=39) and cats (n=36) obtained in Northern Portugal 26
were seeded on Levine agar plates, and two Escherichia coli isolates per sample were 27
recovered (78 of dogs and 66 of cats). The susceptibility to 16 antimicrobial agents was 28
tested in this series of 144 E. coli isolates. Almost 20% of them showed tetracycline 29
resistance and 12 and 15% presented ampicillin or streptomycin resistance, respectively. 30
The percentage of resistance to the other antimicrobial agents was in all cases below 4% 31
and no resistant isolates were detected for ceftazidime, imipenem, cefoxitin or amikacin. 32
Two isolates (from one dog) showed cefotaxime-resistance and harboured both the 33
CTX-M-1 and OXA-30 beta-lactamases. A blaTEM gene was detected in 12 of 17 34
ampicillin-resistant isolates, the aac(3)-II gene in the three gentamicin-resistant isolates, 35
aadA in seven of 22 streptomycin-resistant isolates, and tet(A) and/or tet(B) gene in all 36
28 tetracycline-resistant isolates. The gene encoding class 1 integrase was detected in 37
six E. coli isolates, including the four trimethoprim-sulfamethoxazole-resistant isolates 38
and those two harbouring CTX-M-1 and OXA-30 beta-lactamases; different gene 39
cassette arrangements were identified: dfrA1+aadA1 (2 isolates), dfrA12+orfF+aadA240
(2 isolates) and blaOXA30+ aadA1 (2 isolates). One amino acid change in GyrA protein 41
(Ser83Leu or Asp87Tyr) was detected in four nalidixic-acid-resistant and ciprofloxacin-42
susceptible isolates and two amino acid changes in GyrA (Ser83Leu + Asp87Asn) and 43
one in ParC (Ser80Ile) were identified in one nalidixic acid- and ciprofloxacin-resistant 44
isolate. Faecal E. coli isolates of healthy pets harbour could be a reservoir of 45
antimicrobial resistance genes.46
47
48
49
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1. Introduction50
In the last years there is a great concern about the problem of antimicrobial 51
resistance either in human and in animal medicine, that is associated with failures in the 52
treatment of infectious diseases. The high use of antimicrobial agents in humans and in 53
animals is probably the main cause of this situation (Authier et al., 2006). These agents 54
exert a selection pressure not only on pathogenic bacteria, but also on commensal 55
microorganisms of the intestinal tract of humans and animals, and resistant commensal 56
bacteria constitute a reservoir of resistant genes for potentially pathogenic bacteria (van 57
den Bogaard and Stobberingh, 2000; Guardabassi et al., 2004; Moyaert et al., 2006; de 58
Graef et al., 2004). Escherichia coli is commonly found in the intestinal tract of 59
animals and humans (Tannock, 1995; Sørum and Sunde, 2001), and can also be 60
implicated in animal and human infectious diseases (Sáenz et al., 2001; Rosas et al., 61
2006). For this reason faecal E. coli is considered as a very good indicator for selection 62
pressure by antimicrobial use and for resistance problems to be expected in pathogens 63
(van den Bogaard and Stobberingh, 2000).64
Cats and dogs are companion animals that are in close contact with humans 65
since ancient times, being possible the transference of bacteria between animals and 66
humans (Guardabassi et al., 2004). Various authors have studied antimicrobial 67
resistance in E. coli isolates recovered from pets and these studies have been performed68
either in Europe (Nordman et al., 2000; Guardabassi et al., 2004; Carattoli et al., 2005; 69
Moyaert et al., 2006), as well as in other continents (Authier et al., 2006; Ogeer-Gyles 70
et al., 2006). In Portugal there are studies about antimicrobial resistance in E. coli71
isolates recovered from human clinical samples (Mendonça et al., 2006; Machado et al., 72
2006), healthy humans (Machado et al., 2004), pigs (Pena et al., 2004), and clinical 73
samples of pets (Féria et al., 2002); nevertheless, to our knowledge, there is only one 74
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previous study carried out on healthy pets (Costa et al., 2004), but in that case the 75
unique objective was to detect the presence of E. coli isolates harbouring extended-76
spectrum beta-lactamases. The aim of our present study is to investigate the prevalence 77
of antimicrobial resistances in faecal E. coli isolates recovered from healthy dogs and 78
cats in Portugal and the mechanisms of resistance implicated in order to assess the 79
possible role of the faecal E. coli isolates of pets as a reservoir of antimicrobial 80
resistance.81
82
2. Material and Methods83
Samples and bacterial isolates. Seventy-five faecal samples of healthy pets (39 of 84
dogs and 36 of cats) were included in this study. They were obtained from individually 85
owned animals in two cities of Northern Portugal in 2003, and they were collected 86
either during routine examination of the animals at two veterinary clinics (one located in 87
Porto and the other in Vila Real) or directly by their owners. None of the animals had 88
taken antimicrobials during the four months prior to sampling. All the samples were 89
seeded on Levine agar plates and incubated at 37 °C for 24 h. Two colonies per sample 90
with typical E. coli morphology were selected and identified by classical biochemical 91
methods (gram, catalase, oxidase, indol, Methyl-Red-Voges-Proskauer, citrate and 92
urease), and by the API 20E system (BioMérieux, La Balme Les Grottes, France).93
Antimicrobial susceptibility testing. Antimicrobial susceptibility was performed by 94
the agar disk diffusion method as recommended by the Clinical and Laboratory Standards95
Institute (CLSI, 2007), and a total of 16 antimicrobial agents were tested: ampicillin, 96
amoxicillin-clavulanic acid (AMC), cefotaxime, cefoxitin, ceftazidime, imipenem, 97
aztreonam, gentamicin, tobramycin, amikacin, streptomycin, tetracycline, trimethoprim-98
sulfamethoxazole (SXT), nalidixic acid, ciprofloxacin and chloramphenicol. The 99
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isolates with resistance to one or more antimicrobial agents were selected for the 100
characterization of antimicrobial resistance genes.101
Characterization of antimicrobial resistance genes. The presence of genes encoding 102
TEM, SHV, OXA, CTX-M, and CMY beta-lactamases were studied by PCR in all 103
ampicillin-resistant isolates using primers and conditions previously reported (Table 1). 104
The obtained DNA amplicons were sequenced on both strands and sequences were 105
compared with those included in the GeneBank database in order to identify the specific 106
beta-lactamase gene. In addition, the presence of tet(A), tet(B), tet(C), tet(D) and tet(E) 107
genes were studied by PCR for the tetracycline-resistant isolates. The following genes 108
were also studied by PCR: aadA1 and aadA2 (in streptomycin-resistant isolates),109
aac(3)-I, aac(3)-II and aac(3)-IV (in gentamicin-resistant isolates), and sul1, sul2 and 110
sul3 (in SXT-resistant isolates). The presence of the intI1 and intI2 genes, encoding 111
class 1 and 2 integrases, respectively, as well as qacEΔ1 gene, part of the 3´conserved 112
segment of the class 1 integrons, were also analysed by PCR in SXT-resistant isolates. 113
The variable region of class I integrons was studied by PCR and sequencing. Primers 114
and conditions used for all PCRs are indicated in Table 1. Positive and negative controls 115
from the bacterial collection of the University of La Rioja, Spain, were used in all 116
assays.117
Characterization of the mechanisms of quinolone resistance. The quinolone-118
resistance-determining region (QRDR) of the gyrA gene, as well as the analogous 119
region of the parC gene, were amplified by PCR in all quinolone-resistant E. coli120
isolates (Sáenz et al., 2003). Amplified fragments were purified (Qiagen), and both 121
strands were automatically sequenced by the Applied Biosystem 3730 sequencer 122
(Genome Express, France), using the same set of primers as for the PCR reactions. 123
Sequences obtained were compared with those previously reported for gyrA (GenBank 124
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accession number X06373) and parC genes (M58408 with the modification included in 125
L22025).126
127
3. Results128
A total of 144 E. coli isolates were recovered from the 75 faecal samples of dogs 129
and cats analysed in this study (78 isolates of dogs and 66 of cats). No E. coli isolates 130
were recovered in three of the faecal samples obtained of cats. The susceptibility to 16 131
antimicrobial agents for these isolates is shown in Table 2. Almost 20% of the isolates 132
showed tetracycline resistance and 12-15% of the isolates exhibited ampicillin or 133
streptomycin resistance. The percentage of resistance to the other antimicrobial agents 134
was in all cases below 4% and no resistant isolates were detected to ceftazidime, 135
imipenem, cefoxitin or amikacin. It is interesting to indicate that two isolates (from the 136
same animal) showed cefotaxime and aztreonam resistance. Table 2 shows the 137
percentages of antimicrobial resistance detected depending on the canine or feline origin 138
of the isolates. 139
The phenotypes of resistance exhibited by the 144 E. coli isolates are presented in 140
Table 3. The most frequent detected phenotype was tetracycline-resistance, that was 141
found among 6.3% of the isolates, followed by ampicillin-tetracycline-streptomycin-142
resistance and streptomycin-resistance (3.5% each one). Seventy-two per cent of the 143
E. coli isolates showed a susceptible phenotype to the 16 antimicrobial agents tested. 144
The phenotype of antimicrobial resistance exhibited by the two isolates recovered 145
from the same animal were compared in order to know the degree of diversity among 146
them. In 83% of the animals, the two recovered E. coli isolates presented similar 147
phenotype of resistance, differing in the remaining 17% of the cases.148
149
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The presence of -lactamase genes was investigated in all 17 ampicillin-resistant 150
isolates and a blaTEM gene was detected in 12 of them. Other two of the ampicillin-151
resistant isolates (recovered from one dog) showed cefotaxime and aztreonam resistance 152
and they harboured the genes encoding CTX-M-1 and OXA-30 beta-lactamases. No 153
beta-lactamase genes were identified in the remaining three ampicillin-resistant isolates, 154
which were recovered from samples of cat origin (Table 4). The aac(3)-II gene was 155
identified in the three gentamicin-resistant isolates of cat origin detected in this study,156
and the aadA gene was detected in 7 of 22 streptomycin-resistant isolates (all of them 157
recovered from dogs). In addition, tet(A) and/or tet(B) genes were found in all 28 158
tetracycline-resistant isolates (Table 4). It is interesting to underline that tet(A) was 159
more frequently detected among E. coli isolates of dogs and tet(B) gene among those of 160
cats.161
The intI1 gene encoding class 1 integrase was detected in all four SXT-resistant 162
isolates, but only two of them showed the qacEΔ1 and sul1 genes and amplified the 163
class 1 integron variable region that included in both cases the dfrA1 plus aadA1 gene 164
cassettes arrangement. The remaining two SXT-resistant isolates were studied in detail 165
by PCR mapping and a 1,650 bp amplicon was obtained using the primers Int-F and 166
aadA-R. The sequencing of this fragment revealed the presence of the dfrA12 plus orfF167
plus aadA2 gene cassette arrangement. The sul1 + sul2 or sul3 genes were detected in 168
these SXT-resistant isolates (Table 4). 169
In addition, the intI1 and qacEΔ1 plus sul1 genes were detected in the two E. coli 170
isolates which harbored both CTX-M-1 and OXA-30 beta-lactamases. Their variable 171
regions of class 1 integron were analyzed and the blaOXA30 plus aadA1 gene cassettes 172
were found in both isolates. 173
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The gyrA and parC genes were amplified and sequenced in all five quinolone-174
resistant isolates and the deduced amino acid changes detected in GyrA and ParC 175
proteins are shown in Table 5. Two amino acid changes in GyrA (Ser83Leu + 176
Asp87Asn) and one in ParC (Ser80Ile) were identified in the nalidixic acid- and 177
ciprofloxacin-resistant isolate found in this study and only one amino acid change in 178
GyrA (Ser83Leu or Asp87Tyr) was found in the four nalidixic acid-resistant and 179
ciprofloxacin-susceptible isolates. 180
181
4. Discussion182
The moderate percentages of resistance of the faecal E. coli isolates of 183
healthy pets for ampicillin, streptomycin, and tetracycline (12-19%) identified in our 184
study, are similar to those previously detected in faecal E. coli isolates of cats in 185
Belgium (Moyaert et al., 2006), or in clinical isolates of pets in Switzerland (Lanz et 186
al., 2003). Nevertheless, higher percentages of resistance for these antimicrobial 187
agents (43-50%) were also reported in clinical E. coli isolates of pets in UK by other 188
authors (Normand et al., 2000). In addition, very high percentages of resistance to 189
tetracycline and ampicillin have been detected in faecal isolates of healthy food-190
producing animals (Sáenz et al., 2001). The high use of antimicrobial agents in 191
food-producing animals in relation with healthy pets might explain these 192
differences.193
A TEM beta-lactamase is the most frequent mechanism of ampicillin 194
resistance among our isolates (71%), as it has also been previously detected in 195
ampicillin-resistant E. coli isolates recovered from food, animals and humans 196
(Briñas et al., 2002). It is important to point out the detection in our study of two E. 197
coli isolates, obtained from the same animal (a seven month-old dog), which198
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harboured the genes encoding for CTX-M-1 and OXA-30 beta-lactamases. An E. 199
coli isolate harbouring CTX-M-1 beta-lactamase was previously detected from the200
same animal when its faecal sample was seeded on a Levine agar plate 201
supplemented with 2 mg/L of cefotaxime for the study of faecal colonization by 202
ESBL-containing E. coli (Costa et al., 2004). It seems that the level of colonization 203
by ESBL-containing E. coli isolates was high in this animal because this resistant 204
isolate was also detected in this study when non-supplemented media was used for 205
E. coli detection. As far as we know, this dog had not received antimicrobial agents 206
in the previous four months of sampling, and considering the short life of the animal 207
(seven month old), it might have not received any antimicrobial agent in its whole 208
life. This is the first report, to our knowledge, of an E. coli isolate harbouring both 209
CTX-M-1 and OXA-30 beta-lactamases in animals, and probably also in humans. 210
There are previous reports about E. coli isolates harbouring a CTX-M in addition to 211
an OXA-30 beta-lactamase, but they were obtained in hospitals and the extended-212
espectrum beta-lactamase was CTX-M-15 (Pai et al., 2006; Mendonça et al., 2006; 213
Kim et al., 2005). On the other hand, the presence of the blaOXA-30 plus aadA1 gene 214
cassettes combination inside a class 1 integron variable region has been previously 215
described in E. coli of different origins (Dubois et al., 2003; Sunde, 2005), but this 216
is the first report of E. coli isolates from pets.217
The detection of tet(A) and/or tet(B) genes in all our tetracycline-resistant 218
isolates indicates that the main mechanism of tetracycline resistance in pet E. coli219
isolates is by active efflux. To date, eight different tet genes for efflux proteins have 220
been sequenced in gram-negative bacteria [tet(A-E), (G), (H) and (J)] (Schwarz et 221
al., 2001). A predominance of tet(A) gene has been observed among tetracycline-222
resistant E. coli isolates of dogs, and tet(B) gene among isolates of cats.223
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Four classes of AAC(3) acetyltransferases have been reported associated 224
with gentamicin resistance in E. coli. In our three gentamicin-resistant isolates 225
recovered from cats, the gene encoding AAC(3)-II enzyme was identified. This 226
mechanism of resistance has been also detected in a gentamicin-resistant E. coli227
recovered from a broiler (Sáenz et al., 2004), although the AAC(3)-IV enzyme 228
seems to be more frequent in animal isolates (Guerra et al., 2003).229
It is interesting to point out that the E. coli isolates recovered from healthy 230
pets in this study showed in general low percentages of resistance to 231
aminoglycosides (with the exception of streptomycin), quinolones, chloramphenicol 232
and trimethoprim-sulfamethoxazole, and these values were lower than those 233
previously reported for E. coli from food-producing animals or sick animals (Sáenz 234
et al., 2001; Normand et al., 2000; Lanz et al., 2003; Carattoli et al., 2005).235
The detection of class 1 integrons in some of our E. coli isolates indicates 236
that this genetic mechanism for gene acquisition is present not only among clinical 237
isolates but also in E. coli isolates of the normal microbiota of pets. The 238
combination of two gene cassettes (dfrA1+aadA1) encoding resistance to 239
streptomycin and trimethoprim was identified in two integron-positive isolates. This 240
gene combination has been frequently detected among resistant E. coli isolates of 241
healthy animals and food products (Sáenz et al., 2004; Sunde, 2005). Two additional 242
intI1-positive isolates contained the combination dfrA12 plus orfF plus aadA2 gene 243
cassettes inside the class 1 integron variable region, and lacked the qacEΔ1 and sul1 244
genes on the integron 3’-conserved region which is a non-expected result because 245
the intI1, sul1 and qacEΔ1 genes are usually included in class 1 integrons (Mazel et 246
al., 2000). Nevertheless, this phenomenon has been previously reported and in 247
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addition, it has been associated with this gene cassette arrangement (dfrA12 + orfF + 248
aadA2) (Guerra et al., 2003; Sáenz et al., 2004; Sunde, 2005).249
It has been observed a correlation in the type and number of amino acid 250
changes in GyrA and ParC proteins with the level of resistance to nalidixic acid and 251
ciprofloxacin. This observation has been previously detected either in human E. coli252
isolates and also in animal isolates (Vila et al., 1996; Sáenz et al., 2003).253
As a conclusion, moderate percentages of resistance to ampicillin, 254
streptomycin, and tetracycline and low percentages for the other antimicrobial 255
agents have been detected in faecal E. coli isolates of healthy pets in Portugal. These 256
percentages are in general lower that those previously reported for food-producing 257
animals or from sick pets and could reflect a low antimicrobial pressure in this type 258
of animals in comparison with the other ones. Nevertheless, it is of interest the 259
detection of ESBL-producing E. coli isolates in pets, even in this case in which no 260
supplemented antimicrobial media was used for E. coli selection. More studies 261
should be carried out in the future in order to track the evolution of this type of 262
resistance among the faecal E. coli isolates of different ecosystems.263
264
Acknowledgements265
We thank the Veterinary Hospital Montenegro of Porto (Portugal) for their contribution 266
for the sample collection. This work has been supported in part by Acções Integradas 267
Luso-Espanholas (E-110/06).268
269
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Table 1. Primers and annealing temperatures used in the PCR reactions carried out in this study for detection of antimicrobial resistant
mechanismsa
Primer name Sequence (5’→ 3’) Target gene(s) or region Amplicon
size (bp)
Annealing
temp (°C)
TEM-F ATTCTTGAAGACGAAAGGGC blaTEM 1,150 60
TEM-R ACGCTCAGTGGAACGAAAAC
SHV-F CACTCAAGGATGTATTGTG blaSHV 885 52
SHV-R TTAGCGTTGCCAGTGCTCG
OXA-1 F ACACAATACATATCAACTTCGC blaOXA 813 61
OXA-1 R AGTGTGTTTAGAATGGTGATC
CTX-M-10 F CCGCGCTACACTTTGTGGC blaCTX-M-10 944 52
CTX-M-10 R TTACAAACCGTTGGTGACG
CTX-M-1 group F GTTACAATGTGTGAGAAGCAG blaCTX-M group 1 1,049 50
CTX-M-1 group R CCGTTTCCGCTATTACAAAC
CMY-F GATTCCTTGGACTCTTCAG blaCMY 1,800 53
CMY-R TAAAACCAGGTTCCCAGATAGC
Tet A-F GTAATTCTGAGCACTGTCGC tetA 937 62
Tet A-R CTGTCCTGGACAACATTGCTT
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Tet B-F CTCAGTATTCCAAGCCTTTG tetB 416 57
Tet B-R CTAAGCACTTGTCTCCTGTT
Tet C-F TCTAACAATGCGCTCATCGT tetC 570 62
Tet C-R GGTTGAAGGCTCTCAAGGGC
Tet D-F ATTACACTGCTGGACGCGAT tetD 1,104 57
Tet D-R CTGATCAGCAGACAGATTGC
Tet E-F GTGATGATGGCACTGGTCAT tetE 1,179 62
Tet E-R CTCTGCTGTACATCGCTCTT
AadA-F GCAGCGCAATGACATTCTTG aadA1 or aadA2 282 60
AadA-R ATCCTTCGGCGCGATTTTG
AacC1-F ACCTACTCCCAACATCAGCC aac(3)-I 169 60
AacC1-R ATATAGATCTCACTACGCGC
AacC2-F ACTGTGATGGGATACGCGTC aac(3)-II 237 60
AacC2-R CTCCGTCAGCGTTTCAGCTA
AacC4-F CTTCAGGATGGCAAGTTGGT aac(3)-IV 286 60
AacC4-R TCATCTCGTTCTCCGCTCAT
Sul1-F TGGTGACGGTGTTCGGCATTC sul1 789 63
Sul1-R GCGAGGGTTTCCGAGAAGGTG
Sul2-F CGGCATCGTCAACATAACC sul2 722 50
Sul2-R GTGTGCGGATGAAGTCAG
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Sul3-F GAGCAAGATTTTTGGAATCG sul3 792 51
Sul3-R CATCTGCAGCTAACCTAGGGCTTTGGA
IntI1-F GGGTCAAGGATCTGGATTTCG intI1 483 62
IntI1-R ACATGGGTGTAAATCATCGTC
IntI2-F CACGGATATGCGACAAAAAGGT intI2 788 62
IntI2-R GTAGCAAACGAGTGACGAAATG
Int-F GGCATCCAAGCAGCAAG Class 1 integron variable region variable 55
Int-R AAGCAGACTTGACCTGA
Qac-F GGCTGGCTTTTTCTTGTTATCG qacEΔ1 287 62
Qac-R TGAGCCCCATACCTACAAAGC
GyrA-F TACACCGGTCAACATTGAGG gyrA 648 64
GyrA-R TTAATGATTGCCGCCGTCGG
ParC-F AAACCTGTTCAGCGCCGCATT parC 395 55
ParC-R GTGGTGCCGTTAAGCAAA
a All these primers have been previously included in the following references: Briñas et al., 2003; Coque et al., 2002; Mazel et al., 2000;
Pagani et al., 2003; Sáenz et al., 2004; Sáenz et al., 2003.
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Table 2. Percentages of antimicrobial resistance in the series of 144 E. coli isolates from
faecal samples of pets
Antimicrobial resistant E. coli isolated from:
Dogs (n=78) Cats (n=66) Total pets (n=144)
Antimicrobial
agenta
Number Percentage Number Percentage Number Percentage
Ampicillin 6 7.7 11 16.7 17 11.8
AMCb 2 2.6 3 4.5 5 3.5
Cefotaximeb 2 2.6 0 0 2 1.4
Aztreonamb 2 2.6 0 0 2 1.4
Ceftazidime 0 0 0 0 0 0
Cefoxitine 0 0 0 0 0 0
Imipenem 0 0 0 0 0 0
Gentamicin 0 0 3 4.5 3 2.1
Tobramycin 0 0 2 3.0 2 1.4
Amikacin 0 0 0 0 0 0
Streptomycin 14 17.9 8 12.1 22 15.2
Tetracycline 16 20.5 12 18.2 28 19.4
SXT 4 5.1 0 0 4 2.8
Nalidixic acid 3 3.8 2 3.0 5 3.5
Ciprofloxacin 1 1.3 0 0 1 0.7
Chloramphenicol 4 5.1 0 0 4 2.8
aAMC, amoxicillin-clavulanic acid; SXT, trimethoprim-sulfamethoxazole.
bIsolates in the resistant and intermediate category are included in this section
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Table 3. Phenotypes of resistance detected among the 144 E. coli isolates recovered
from pets.
Phenotype of resistancea Number of isolates Percentage of isolates
TET 9 6.3
STR 5 3.5
NAL 2 1.4
TET-STR 3 2.1
TET-NAL 2 1.4
AMP-TET 1 0.7
AMP-GEN 1 0.7
AMP-STR-SXT 2 1.4
AMP-GEN-TOB 2 1.4
AMP-TET-STR 5 3.5
TET-STR-SXT-CHL 2 1.4
AMP-AMCb-STR-TET 3 2.1
AMP-TET-NAL-CIP 1 0.7
AMP-AMCb-CTXb-ATMb-STR-TET-CHL 2 1.4
Susceptible 104 72.2
aAMP, ampicillin; AMC, amoxicillin-clavulanic acid; CTX, cefotaxime; ATM,
aztreonam; GEN, gentamicin; TOB, tobramycin; STR, streptomycin; TET, tetracycline;
SXT, trimethoprim-sulfamethoxazole; NAL, nalidixic acid; CIP, ciprofloxacin; CHL,
chloramphenicol.bResistance to the drug indicated is in the intermediate or resistance category according
to CLSI standards.
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Table 4. Genes of resistance detected among our antimicrobial resistant E. coli isolates of dog and cat origins.
Dogs Cats
Genes detected Genes detected
Phenotype of
resistanceNumber of isolates
with this phenotype Genes Number of isolates
No. of isolates with
this phenotype Genes Number of isolates
Ampicillin 6 blaTEM
blaCTX-M-1 +blaOXA-30
4
2a
11 blaTEM 8
Gentamicin 0 - - 3b aac(3)-II 3
Streptomycin 14 aadA 7 8 - -
Tetracycline 16 tet(A)
tet(B)
tet(A)+tet(B)
10
5
1
12 tet(A)
tet(B)
1
11
SXT c 4 dfrA1d + sul1+ sul2
dfrA12d + sul3
2
2
0 - -
a These isolates showed also resistance to cefotaxime and aztreonamb Two of these three isolates showed also tobramycin resistancecSXT: Trimethoprim-sulfamethoxazoledThis gene was found inside a class 1 integron
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Table 5. Amino acid changes in GyrA and ParC proteins deduced from the sequences
of the corresponding genes in our quinolone-resistant E. coli isolatesa
Phenotype of resistance
to quinolonesa
Number of E. coliisolates
Amino acid changes in:
GyrA ParC
Nalidixic acid-ciprofloxacin 1 Ser83Leu + Asp87Asn Ser80Ile
Nalidixic acid 2 Asp87Tyr wild
Nalidixic acid 2 Ser83Leu wild
aSequences were compared with gyrA and parC genes included in the GenBank
database with the accession numbers X06373 for gyrA and M58408 with the
modification in L22025 for parC.
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